This report describes pyrimidine oligonucleotides (ONs) designed to complement a specific target on the HIV-1 proviral genome and form a "triple helix" structure which could affect virus activity by blocking the major groove of the target double-stranded DNA. We designed several ONs complementary to purine-rich regions within the HIV-1 genome with the goal of suppressing HIV infection. The HIV genes targeted by these ONs include gag, pol, tat, nef and vif. We tested the ability of these ONs to enter the cells and suppress HIV-1 (MN) infection of the H9 lymphocyte cell line. ON and virus were added directly to cell media at the same time. ONs were present in the nuclei of treated cells within 10 mins of initial treatment. Treatment of infected cells with 10 to 50 uM concentrations of any of the ONs significantly reduced virus levels as demonstrated by: 1) a 50-90% decrease in extracellular p24 antigen as measured by p24 capture ELISA, 2) a concomitant decrease in intracellular p24 as detected by immunofluorescence labeling and FACSCAN analysis, 3) a similar reduction in amounts of viral DNA (detected by DNA PCR assay of cell lysates) relative to untreated infected cells. Cell growth and viability was not affected at these low ON concentrations. In an initial study, infected cells treated with 50 uM of GAG specific ON produced 85% less p24 antigen than infected cells treated with 50 uM of a nonspecific, pyrimidine ON with similar base composition (control). Further studies with nonspecific ONs are in progress. Preliminary studies with primary human peripheral blood lymphocytes also demonstrated suppression of virus by HIV-specific triple helix ONs. These ONs show promise as anti-HIV agents. Ongoing studies are addressing 1) the effects of ON treatment on viral transcripts, 2) the persistence of ON in cells and nuclei, and 3) the ability of the ONs to form triple helix complexes with HIV sequences.